Field of the Invention
The invention concerns methods helping the manual deposition of samples, preferably of biological material, on a mass spectrometric sample support for ionization by laser desorption, preferably by matrix assisted laser desorption, and corresponding deposition aids.
Description of the Related Art
The rapid, error-free identification of microorganisms plays a prominent role in the analysis of food, in the monitoring and control of biotechnological processes, in the detection of biological weapons and particularly in clinical microbiology. Microorganisms, which are also called germs or microbes, are usually microscopically small living organisms which include bacteria, fungi (e.g. yeasts), microscopic algae, protozoa—for example plasmodia, which cause malaria—and in some sense also viruses.
The identification of bacteria by mass spectrometric detection methods has been described in detail in a scientific review article by van Baar, for example (FEMS Microbiology Reviews, 24, 2000, 193-219: “Characterization of bacteria by matrix-assisted laser desorption/ionization and electrospray mass spectrometry”). In most cases the identification is achieved by means of a similarity analysis between a mass spectrum of the sample under investigation and reference spectra of known microorganisms. The similarity analysis involves assigning each reference spectrum a similarity indicator which is a measure of the agreement between the relevant reference spectrum and the mass spectrum of the sample (see for example Jarman et al., Analytical Chemistry, 72[6], 1217-1223, 2000: “An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry”).
In recent years, this simple and low-cost method for the mass-spectrometric identification of microorganisms based on MALDI time-of-flight mass spectra (MALDI=matrix assisted laser desorption and ionization) has become established for routine work in clinical microbiology.
The starting point for the mass spectrometric identification is a small quantity of microorganisms, which are usually cultured in a culture dish, such as a Petri dish with a nutrient medium (agar plate), for some hours—usually overnight culture—up to a few days. The aim is that the colonies grown in the agar plate each contain only species of one single microorganism, i.e. they form a pure culture. The usual method of sample preparation is to manually take up biological material of a single colony on an agar plate with an inoculation swab, a type of toothpick, for example, and transfer it to a sample site on a MALDI sample support. Conventional MALDI sample supports have between 6 and 1536, in particular 48 to 384 sample sites. If the quantity of sample transferred with the inoculation swab can just be seen with the naked eye, it is already dosed a little high for a mass spectrometric investigation. One million microbes in the sample makes the sample just visible; the optimum number for excellent mass spectra amounts to some 100,000 microbes.
The transferred cells are usually destroyed by the addition of an organic solvent in which a matrix substance is dissolved. This releases molecular cell components from the inside of the cell, in particular soluble proteins which are present in high concentration. The organic solvent evaporates during air-drying and the matrix substance crystallizes. The molecular cell components released in this process are incorporated into the polycrystalline matrix layer. New inoculation swabs are used for the preparation of further sample sites on the MALDI sample support in order to prevent cross-contamination.
After the preparation, the MALDI sample support is introduced into a MALDI time-of-flight mass spectrometer, where the sample sites are bombarded with laser pulses. In this way, the molecular cell components embedded in the matrix layer are desorbed and ionized together with the matrix substance. The ions are accelerated in an electric field and impact on a detector after mass-dependent times of flight. The times of flight of the ions measured with the detector are converted into mass-to-charge ratios m/z with the aid of known mass calibration functions. The measured signals can often be traced back to proteins which are specific for the species of the microorganism and sometimes even for the strain. The mass spectrum can thus be interpreted as a molecular fingerprint and can be used for microbial identification in particular.
The publication DE 10 2004 020 885 A1 is concerned with the preparation of samples of microbial origin on MALDI sample supports with the objective of automating the transfer of biological materials from agar plates to sample sites on MALDI sample supports. To this end, agar plates are transported via a conveyor belt to a robot and set down on a 3D stage. An image processing system recognizes separated colonies on the agar plate and positions a sampling rod accordingly. An individual sampling rod is used for one single transfer only and replaced afterwards. Biological materials are taken up by the sampling rod being released from a holder and dropping from a height of a few millimeters onto the colony. The contact with the colony thus achieved is intended to ensure that only biological material adheres to the sampling rod, and no agar is transferred onto the MALDI sample support. If too much agar is transferred onto the MALDI sample support, the quality of the mass spectrometric identification is reduced because agar suppresses the signals of the characteristic protein ions. A high-precision sensor system to control the contact is not provided. The sampling rod does, however, vibrate, and it can be wetted with water before the sampling in order that a sufficient quantity of biological material from a colony adheres to the sampling rod and can be transferred onto a sample site of a MALDI sample support.
In a semi-automatic sampling system, a single colony from which biological material is to be transferred onto a MALDI sample support is selected by a user selecting and marking the single colonies on an image of the agar plate taken by a camera, for example, before the automatic transfer.
The advantage of an automatic and semi-automatic transfer from an agar plate onto a MALDI sample support consists in the agar plate and the sampling location on the agar plate being uniquely assigned to the sample sites of a MALDI sample support, and samples of microbial origin from different colonies being transferred to one single sample site, thus preventing any mix-up. If, in addition, an image of the sampling on the agar plate is acquired and stored, even an individual colony can be uniquely assigned to a sample site. The agar plates and the MALDI sample supports are nowadays usually provided with corresponding codings, such as barcodes or RFID chips (RFID=radio frequency identification). The journey of a sample from its arrival in the laboratory—or even from it being taken at the doctor's office—through to the acquisition and evaluation of the mass spectra can therefore be traced back in an unbroken chain.
With manual transfer, on the other hand, transfer mistakes can easily happen, with the result that several samples of microbial origin are transferred onto one sample site as a result of a mistake by the laboratory staff, or the assignment of samples on the MALDI sample support is incorrectly recorded in a laboratory information and management system. In these cases the identification of the microorganisms is also erroneous, of course. The automation of the sample transfer has gained very little acceptance so far, however, because the mass spectra of manually prepared samples have a higher quality throughout. There is therefore a need to improve the methods of manual preparation.
The utility model DE 20 2007 018 535 U1 describes a pipetting aid for transparent microtiter plates, which are put into a base plate with the aid of an adapter. The base plate contains sources of light, which are each assigned to an opening in the adapter and a cavity of the transparent microtiter plate. A switching or control unit activates the light sources independently of each other, and the light passing through the adapter and the transparent microtiter plate indicates where a sample liquid is to be pipetted. In contrast to the microtiter plates, sample supports for ionization with matrix assisted laser desorption are generally opaque. This is usually a result of their electrical conductivity, which serves to prevent static charges on the sample support, which can form during the laser desorption. Electrical conductivity is fundamentally undesirable for microtiter plates because the cavities—in contrast to the flat sample sites on the MALDI sample supports, which are, to a large extent, designed flush with the rest of the surface—provide a larger interaction area with the pipetted sample liquid. This enlarged interaction area can—if the plate is conductive and the samples are liquid—promote undesirable boundary layer processes, for example the deposition of charge carriers which are dissolved in the liquid, such as salts, or chemical boundary layer reactions.
It is relatively easy for a lab technician who has transferred sample liquid onto a microtiter plate to recognize which cavities contain the sample liquid and which do not. This particularly results from the fact that, for manual pipetting, at least one microliter of sample liquid has to be transferred because it is not usually possible to precisely measure a smaller amount with the pipettes used. The quantity of one microliter is sufficiently large that a person can recognize it with certainty with the naked eye, which is also guided by the cavities themselves to some degree.
When a flat MALDI sample support plate is spotted with microorganisms, it is hardly possible to recognize correctly dosed samples with the eye. In laboratory practice, one occasionally resorts to picking up the sample support in the hand after applying a sample and holding it against the light to make out a sample on the sample site. Only if the matrix substance with solvent is applied to a sample site immediately after applying the analyte substance of interest, such as biological materials from a cultured microbe colony, can the lab technician clearly recognize the distribution of the samples on the sample support at a glance and without much effort because the light reflection and scattering properties of the liquid spot—or the matrix crystal layer produced on the sample site after this liquid has evaporated—differ from those of the rest of the sample support surface. However, this procedure of applying the matrix solution immediately makes the spotting sequence less flexible, which a lab technician usually finds unhelpful. In addition, confusion can occur if, for example, the technician is distracted during the spotting sequence and forgets to apply matrix substance with solvent onto a sample site previously spotted with an analyte substance.
As a result of these considerations it would be expedient to, in particular, reduce the risk of an incorrect assignment of the samples to the sample sites during the manual depositing and preparation of samples for ionization with matrix assisted laser desorption.